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Simple Summary: In this study, we aimed to improve the culture system for chicken embryonic-derived pluripotent stem cells (PSCs). These cells have immense potential in various fields, such as growth and development studies, vaccine production, and preserving genetic resources. However, establishing a stable and efficient culture system for chicken PSCs remains a challenge. We investigated the effects of different signaling pathways and feeder layers on the derivation and maintenance of chicken PSCs. Our results show that using STO cells as feeder layers, along with specific signaling pathway inhibitors, allows for the efficient derivation of chicken PSC-like cells. These cells exhibit characteristics of pluripotency and can differentiate into various cell types. This research enhances our understanding of chicken PSC culture conditions and lays the groundwork for their biomedical and biotechnological applications. The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal–Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs. [ABSTRACT FROM AUTHOR]

Spindlin 1 (SPIN1), which contains Tudor‐like domains, regulates maternal transcripts via interaction with a messenger RNA (mRNA)‐binding protein. SPIN1 is involved in tumorigenesis in somatic cells and is highly expressed in cancer cells. Nevertheless, the role of SPIN1 in porcine oocyte maturation remains totally unknown. To explore the function of SPIN1 in porcine oocyte maturation, knockdown, and overexpression techniques were used. SPIN1 mRNA was identified in maternal stages ranging from GV to MII. SPIN1 was localized in the cytoplasm and to chromosomes during meiosis. SPIN1 knockdown accelerated first polar body extrusion. Oocytes with overexpressed SPIN1 were arrested at the MI stage. SPIN1 depletion caused meiotic spindle defects and chromosome instability. The BUB3 signal was investigated, confirming that SPIN1 affects the stability of Bub3 mRNA as well as BUB3 expression. Further, overexpression of SPIN1 inhibited the degradation and regulation of G2/mitotic‐specific cyclin‐B1. In summation, SPIN1 regulates the meiotic cell cycle by modulating the activation of the spindle assembly checkpoint. Spindlin 1 (SPIN1) is an important gene for the first meiotic division of porcine oocytes. Oocytes were arrested at the MI stage following SPIN1 overexpression, inducing a longer BUB3 poly (A) tail length and the subsequent high translation of BUB3, further preventing the decrease of activity of CDK1. However, SPIN1 knockdown accelerated the first polar body extrusion with abnormal spindle formation and chromosome segregation. [ABSTRACT FROM AUTHOR]

Fatty acid synthase (FAS) is an important enzyme involved in the de novo synthesis of long‐chain fatty acids. During development, the function of FAS in growth is greater than that in energy storage pathways; therefore, we hypothesized that knockout of FAS would affect early embryonic development owing to the induction of endoplasmic reticulum (ER) stress. In the present study, the function of FAS was studied using the CRISPR (clustered regularly interspaced short palindromic repeats)/ CRISPR‐associated protein 9 (Cas9) system. Cas9 and single‐guide RNA (sgRNA) were injected into parthenotes to decrease the number of FAS‐positive embryos. The efficiency of knockout was assayed by DNA sequencing. We found that FAS knockout caused excessive production of reactive oxygen species (ROS). Excess ROS induced ER stress, resulting in activation of the adaptive unfolded protein response (UPR). FAS knockout caused splicing of the X‐box binding protein 1 gene (XBP1) and expression of spliced XBP1 mRNA. In addition, FAS knockout caused phosphorylation of PKR‐like ER kinase (PERK), and an increase in the mRNA expression of the ER stress‐regulated genes, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). Finally, Ca2+ was released from the ER and taken up by the mitochondria. As the ER stress became intolerable, apoptosis was initiated. These results demonstrate that FAS knockout induced ROS generation, which mediated the activation of UPR via the ER stress, ultimately leading to apoptosis induction. [ABSTRACT FROM AUTHOR]

Melatonin, a major hormone of the pineal gland, exerts many beneficial effects on mitochondria. Several studies have shown that melatonin can protect against toxin‐induced oocyte quality impairment during maturation. However, there is little information regarding the beneficial effects of melatonin on toxin‐exposed early embryos, and the mechanisms underlying such effects have not been determined. Rotenone, a chemical widely used in agriculture, induces mitochondrial toxicity, therefore, damaging the reproductive system, impairing oocyte maturation, ovulation, and fertilization. We investigated whether melatonin attenuated rotenone exposure‐induced impairment of embryo development by its mitochondrial protection effect. Activated oocytes were randomly assigned to four groups: the control, melatonin treatment, rotenone‐exposed, and "rotenone + melatonin" groups. Treatment with melatonin abrogated rotenone‐induced impairment of embryo development, mitochondrial dysfunction, and ATP deficiency, and significantly decreased oxidative stress and apoptosis. Melatonin also increased SIRT1 and PGC‐1α expression, which promoted mitochondrial biogenesis. SIRT1 knockdown or pharmacological inhibition abolished melatonin's ability to revert rotenone‐induced impairment. Thus, melatonin rescued rotenone‐induced impairment of embryo development by reducing ROS production and promoting mitochondrial biogenesis. This study shows that melatonin rescues toxin‐induced impairment of early porcine embryo development by promoting mitochondrial biogenesis. [ABSTRACT FROM AUTHOR]

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV , SOX2 , and NANOG , as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal-Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3 -edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1 ) expression could rescue AGS3 -overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Phosphatase and tensin homolog-induced kinase 1 (PINK1) on the outer membranes of impaired mitochondria promotes mitophagy and regulates mitochondrial morphology. Mammalian oocytes and early embryos are mitochondria rich, but mitochondrial dynamics during preimplantation embryo development is not well-studied. To investigate whether PINK1 is required for mitochondrial dynamics in porcine preimplantation embryos, gene knockdown and inhibitors were used, and mitochondrial dynamics were observed by transmission electron microscopy. PINK1 knockdown significantly impaired blastocyst formation and quality, induced mitochondrial elongation and swelling, and reduced mitochondrial DNA copy number. PINK1 knockdown-induced mitochondrial elongation caused mitochondrial dysfunction, oxidative stress, and ATP deficiency, significantly increasing autophagy and apoptosis. Profission dynamin-related protein 1 overexpression prevented PINK1 knockdown-induced impairment of embryo development, mitochondrial elongation, and dysfunction. Thus, PINK1 promotes mitochondrial fission in porcine preimplantation embryos.-Niu, Y.-J., Nie, Z.-W., Shin, K.-T., Zhou, W., Cui, X.-S. PINK1 regulates mitochondrial morphology via promoting mitochondrial fission in porcine preimplantation embryos.